期刊
BIOCHEMICAL JOURNAL
卷 388, 期 -, 页码 913-920出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20041739
关键词
cis-regulatory element; electrophoretic mobility-shift assay; gene promoter; gene regulation; transcription factor; vascular endothelial growth inhibitor
资金
- NINDS NIH HHS [R01 NS51625, K08 NS002190, R01 NS051625] Funding Source: Medline
VEGI (vascular endothelial growth inhibitor), a member of the tumour necrosis factor superfamily, has been reported to inhibit endothelial cell proliferation, angiogenesis and tumour growth. We identified and cloned approx. 2.2 kb of the VEGI promoter from mouse cerebral endothelial cells. The promoter contained an atypical TATA-box-binding protein sequence TAAAAAA residing at -32/-26 relative to the transcription initiation site (+1), 83 bp upstream from the ATG start codon. To investigate critical sequences in the VEGI promoter, a series of deleted and truncated segments were constructed from a 2300 bp promoter construct (-2201/+96) linked to a luciferase reporter gene. Transient transfection of cerebral microvascular cells (bEND.3) and rat C6 glioma cells demonstrated that a 1700 bp deletion from the -2201 to -501 did not significantly affect promoter activity; however, a truncated construct (-501/+96) lacking the region between -312 and -57 resulted in nearly 90% loss of promoter activity. A consensus NF-kappa B (nuclear factor kappa B) and several SP1 (specificity protein-1)-binding sequences were identified within the deleted segment. Supershift analysis revealed that NF-kappa B subunits, p50 and p65, interacted with the VEGI promoter. Exposure of cerebral endothermic cells to the proinflammatory cytokine, tumour necrosis factor-alpha, increased VEGI mRNA levels and DNA-binding activities, whereas an NF-kappa B inhibitor attenuated this increase. In addition, p65 overexpression enhanced, whereas p50 overexpression decreased, the luciferase activity, Furthermore, mutation of the NF-kappa B DNA binding site blocked this p65- and turnout necrosis factor-alpha-induced luciferase activity. These findings suggest that the transcription factor NF-kappa B plays an important role in the regulation of VEGI expression.
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